The Immune System: 2

Overview of the Immune System by Dr. Sidgi Hasson

• Instructor Information:     * Dr. Sidgi Hasson, Senior Lecturer in Immunology.     * Affiliation: School of Pharmacy and Biomolecular Sciences, LJMU (Liverpool John Moores University).     * Office: James Parsons Building, Room #802.

• Document Scope: This guide serves as a basic overview of the mechanisms of the immune system (Part 2 of 2).

• Primary Classification of Innate System Cells:     * Phagocytes.     * Antigen Presenting Cells (APCs).     * Direct Attack Cells.

• Foundational Concept: Recognition of Self from Non-self.

Fundamental Features of Innate Immune Recognition

• Recognition Specificity: Innate immunity is characterized by "Limited specificity." It recognizes structures and motifs that characterize microbial pathogens but are absent in mammalian cells.

• Pathogen-Associated Molecular Patterns (PAMPs): Conserved structures located on or within microbes. Examples include:     * Cell wall peptidoglycans.     * Lipoteichoic acid (LTA).     * Mannans.     * Lipopolysaccharides (LPS).

• Pattern Recognition Receptors (PRRs): These are receptors of the innate immune system specifically designed to bind PAMPs.

• Toll-Like Receptors (TLRs) Distribution and Specificity:     * Plasma Membrane Receptors:         * $TLR-1:TLR-2$ heterodimer: Recognizes bacterial lipopeptides.         * $TLR-2$: Recognizes bacterial peptidoglycan.         * $TLR-4$: Recognizes Lipopolysaccharides (LPS).         * $TLR-5$: Recognizes bacterial flagellin.         * $TLR-2:TLR-6$ heterodimer: Recognizes bacterial lipopeptides.     * Endosomal Receptors (Intracellular):         * $TLR-3$: Recognizes double-stranded RNA (dsRNA).         * $TLR-7$: Recognizes single-stranded RNA (ssRNA).         * $TLR-8$: Recognizes single-stranded RNA (ssRNA).         * $TLR-9$: Recognizes CpG DNA.     * Cytosolic Receptors: Includes RIG1.

Functional Classification and Surface Receptors of Macrophages

• Macrophage Receptors: Macrophages express multiple receptors to identify bacterial constituents:     * Mannose receptor.     * LPS receptor ($CD14$).     * $TLR-4$.     * Glucan receptor.     * Scavenger receptor.

• Outcome of Binding:     1. Release of Mediators: Initiates the release of cytokines and small lipid mediators of inflammation.     2. Phagocytosis: Macrophages engulf and digest the bacteria following binding.

Mechanisms of Phagocytosis and Intracellular Killing

• Phagocytosis by Neutrophils and Macrophages:     * Killing Mechanism: Primarily through activated proteolytic enzymes and oxygen/nitrogen intermediates.     * Respiratory Burst (Oxidative Burst): Mediated by $NADPH \, Oxidase$. Produces reactive components:         * Hydrogen peroxide ($H_2O_2$).         * Superoxide anion ($O_2^-$).         * Nitric oxide ($NO$).     * Localization: Killing activity is restricted to phagolysosomes to prevent host tissue damage.

• Host Protective Enzymes: These enzymes are produced by phagocytes to protect the host cells from the respiratory burst products:     * Catalase: Degrades $H_2O_2$.     * Superoxide Dismutase: Converts $O_2^-$ into $H_2O_2$ and oxygen.

• The Five Stages of Phagocytosis:     1. Pathogen Recognition: Microbes bind to phagocyte receptors (e.g., Mannose receptor, $Mac-1 \, integrin$, Scavenger receptor).     2. Attachment and Zipping: The phagocyte membrane zips up and surrounds the microbe.     3. Ingestion: The microbe is internalized into a phagosome.     4. Fusion: The phagosome fuses with a lysosome (containing enzymes) to form a phagolysosome.     5. Phagocyte Activation: Killing of the microbe via intracellular mechanisms.

• Intracellular Killing Categories:     * Oxygen-Independent: Use of lysosomal enzymes.     * Oxygen-Dependent: Use of the oxidative (respiratory) burst.

Antigen Presenting Cells (APCs)

• Professional APCs: Dendritic cells, Macrophages (differentiated from monocytes), and B cells.

• Functional Mechanism:     * Partial digestion of protein antigens into peptides.     * Loading of peptides into Major Histocompatibility Complex (MHC) molecules on the cell surface.     * Presentation of the $MHC + peptide$ complex to T cells (initiating adaptive immunity).

• Comparison of Professional APCs:     * Dendritic Cells:         * Antigen Uptake: High ($+++$) via macropinocytosis and phagocytosis by tissue-resident cells.         * MHC Expression: Low on tissue-resident cells; High on cells in lymphoid tissues.         * Co-stimulator Delivery: Constitutive by mature, non-phagocytic lymphoid dendritic cells.         * Primary Antigen presented: Viral antigens and allergens.         * Location: Ubiquitous throughout the body.     * Macrophages:         * Antigen Uptake: High ($+++$) via phagocytosis.         * MHC Expression: Inducible by bacteria and cytokines.         * Co-stimulator Delivery: Inducible ($-$ to $+++$).         * Primary Antigen presented: Particulate antigens; intracellular and extracellular pathogens.         * Location: Lymphoid tissue, connective tissue, body cavities.     * B Cells:         * Antigen Uptake: High ($++++$) via antigen-specific receptors (immunoglobulin/Ig).         * MHC Expression: Constitutive; increases upon activation.         * Co-stimulator Delivery: Inducible ($-$ to $+++$).         * Primary Antigen presented: Soluble antigens, toxins, viruses.         * Location: Lymphoid tissue, peripheral blood.

Direct Attack Cells and Natural Killer (NK) Cells

• Cell Lineages:     * Myeloid Origin: Monocytes, Macrophages, Granulocytes (Neutrophils, Eosinophils, Mast cells, Basophils), and Dendritic Cells.     * Lymphoid Origin: Natural Killer (NK) cells and NK/T cells.

• Cytoplasmic Granules: Direct attack cells contain granules with toxic mediators: histamine, prostaglandins, leukotrienes, tryptase, and eosinophil peroxidase.

• Natural Killer (NK) Cells:     * Status: Ready to kill; do not require activation.     * Killing Mechanisms (Same as Cytotoxic T-cells):         * Perforin: Inserts pores into the target cell membrane.         * Granzymes: Proteolytic enzymes that enter through perforin pores to induce apoptosis/chromosomal degradation.         * FasL/Fas Interaction: Induces apoptosis in target cells.

• Regulation of NK Activity:     * Inhibitory Receptors: Bind to standard surface markers (e.g., $MHC-I$). If $MHC-I$ is present, the "kill signal" is suppressed.     * Activating Receptors: Bind to stress-induced glycoproteins on infected or tumor cells. If $MHC-I$ is absent or low, the activating signal triggers killing.

• Antibody-Dependent Cellular Cytotoxicity (ADCC):     * The $Fab$ portion of an antibody binds to epitopes on a foreign cell.     * The NK cell binds to the $Fc$ portion of that antibody.     * The NK cell releases perforins and granzymes to destroy the cell's cytoskeleton and induce apoptosis.

Lymphocyte Activation, Homing, and Resolution

• B and T Cell Activation Cycle:     1. Naïve Lymphocyte: Encounters antigen + second signals.     2. Lymphoblast: Conversion to a large lymphocyte cell.     3. Clonal Expansion: Proliferation and differentiation.     4. Effector Cells: B cells become antibody-secreting plasma cells; T cells become cytokine-producing cells or CTLs.     5. Memory Cells: Formation of Memory B and T cells for long-term immunity.     6. Homeostasis: Followed by apoptosis of effector cells once the threat is resolved.

• Activation and Homing Process:     * Microbes or antigens enter through the epithelium into connective tissue.     * Dendritic cells capture the antigen and transport it via the afferent lymphatic vessel to a regional lymph node.     * Circulating naïve lymphocytes enter the lymph node through arteries.     * Activation and differentiation occur within the lymph node.     * Effector cells and antibodies exit via the efferent lymphatic vessel and enter general circulation to migrate back to the site of infection.     * Memory lymphocytes take up residence in normal tissues for future protection.

Cytokines, Chemokines, and the Inflammatory Response

• Inflammatory Process:     * Bacteria trigger macrophages to release cytokines and chemokines.     * Vasodilation: Increased vascular permeability leads to redness, heat, and swelling (edema).     * Migration: Inflammatory cells (e.g., neutrophils) migrate into tissue, causing pain through the release of mediators.

• Specific Cytokines Secreted by Macrophages and Dendritic Cells:     * $IL-1$: Produced by macrophages/keratinocytes; acts on lymphocytes (enhances response) and the liver (induces acute-phase proteins).     * $IL-6$: Produced by macrophages/dendritic cells; acts on lymphocytes and the liver (acute-phase protein secretion).     * $CXCL8$ ($IL-8$): Produced by macrophages/dendritic cells; acts as a chemoattractant for neutrophils.     * $IL-12$: Produced by macrophages/dendritic cells; acts on naïve T cells to divert immune response to Type 1 proinflammatory secretion.     * $TNF-\alpha$: Produced by macrophages/dendritic cells; acts on vascular endothelium to increase fluid loss and cell-adhesion molecules ($E-$ and $P-selectin$).

• Phagocyte Bactericidal Products:     * Acidification: $pH \approx 3.5 - 4.0$ (bacteriostatic/bactericidal).     * Toxic Oxygen: Superoxide ($O_2^-$), $H_2O_2$, singlet oxygen ($1O_2$), hydroxyl radical ($OH^\cdot$), hypohalite ($OCl^-$).     * Toxic Nitrogen: Nitric oxide ($NO$).     * Antimicrobial Peptides: Defensins and cationic proteins.     * Enzymes: Lysozyme (dissolves Gram-positive walls), Acid hydrolases.     * Competitors: Lactoferrin (binds Fe), Vitamin $B_{12}$-binding protein.

Comparison: Innate vs. Adaptive Immunity

Questions & Discussion

• The lecture concludes with a placeholder for student questions involving concepts such as B-cells, viruses, and complex immune interactions.

Introduction to Immunology and Immunity

• Etymology and Definitions     - The word "Immunology" originates from the Latin words Immunitas and Immunes.     - Historical Context in Rome: Initially, these words implied the "Exemption of an Individual from Service or Duty."     - Middle Ages Evolution: The term evolved to mean "Exemption of the Church and its properties and personnel from Civil control."     - Modern Definition of Immunity: The ability of an individual to resist diseases.     - Immunochemistry: Coined by Marcus Annaeus Lucanus, this was the initial term for the study of immune reactions.     - Experimental Discipline: Immunology is defined as an experimental discipline that manipulates the function of the immune system.

• History of Immunity Research     - Discovery (1882): Élie Metchnikoff initiated immunity research through an experiment using starfish larvae.     - The Experiment: Metchnikoff poured carmine dye on starfish larvae and observed cells that appeared to ingest the dye.     - Findings: Metchnikoff believed he had found the answer to inflammation, suggesting that specialist cells form a protective mechanism against foreign microbes.     - Phagocytosis: This process of ingestion by cells was later termed phagocytosis.

Roles and Functions of the Immune System

• Primary Roles     - Defense against Microbes: Protecting the body from pathogenic organisms.     - Defense against Tumor Cells: Identifying and killing the growth of tumor cells.     - Homeostasis: The destruction of abnormal or dead cells, such as dead red blood cells (erythrocytes), dead white blood cells (leukocytes), and antigen-antibody complexes.

• Major Functions of Innate Immunity     - Immediate Response: The initial prevention, control, or elimination of infection until adaptive immune responses are activated.     - Increased Susceptibility: Inhibiting or eliminating any component of innate immunity significantly increases susceptibility to infections, even if the adaptive immune system remains intact.     - Stimulation: The innate system stimulates adaptive immune responses to be optimally effective against different types of microbes (e.g., distinguishing between bacteria and viruses).

Blood Cell Composition and Counts

• Normal Cell Counts (per $mm^3$ and percentage of total leukocytes)     - Red Blood Cells (Erythrocytes): $5.0 \times 10^{6}\,cells/mm^3$     - Platelets: $2.5 \times 10^5\,cells/mm^3$     - Leukocytes (Total population distribution):         - Neutrophils: $7.3 \times 10^3\,cells/mm^3$, comprising $50-70\%$ of leukocytes.         - Lymphocytes: Comprising $20-40\%$ of leukocytes.         - Monocytes: Comprising $1-6\%$ of leukocytes.         - Eosinophils: Comprising $1-3\%$ of leukocytes.         - Basophils: Comprising <1\% of leukocytes.

Haematopoiesis: The Development of Immune Cells

• The Process and Lineages     - Pluripotent Hematopoietic Stem Cell: Located in the bone marrow, this is the origin of all blood cells.     - Common Lymphoid Progenitor: Leads to the development of B cells, T cells, and Natural Killer (NK) cells.     - Common Myeloid Progenitor: Leads to the development of granulocyte/macrophage progenitors and megakaryocyte/erythrocyte progenitors.     - Megakaryocyte/Erythrocyte Pathway:         - Megakaryocytes $\rightarrow$ Platelets.         - Erythroblasts $\rightarrow$ Erythrocytes.     - Granulocyte/Macrophage Pathway:         - Neutrophils.         - Eosinophils.         - Basophils.         - Monocytes (precursors of macrophages).         - Unknown precursors lead to mast cells.         - Dendritic cells (can originate from both myeloid and lymphoid precursors).

• Tissue Maturation and Effector Cells     - B Cells: Mature in the bone marrow and differentiate into plasma cells in the tissues.     - T Cells: Mature in the thymus and become activated T cells in the tissues.     - NK Cells: Differentiate into activated NK cells.     - Monocytes: Circulate in the blood and migrate to tissues to become macrophages.

Innate Immunity Framework

• Lines of Defense     - Physical and Chemical Innate Defense: Anatomical and physiological barriers.     - Cellular Innate Defense: Specific cell types responding to invasion.

• A: External Innate Defense (Anatomical Barriers)     - Skin:         - Mechanical: Epithelial cells joined by tight junctions; longitudinal flow of air or fluid.         - Chemical: Fatty acids; antibacterial peptides.         - Microbiological: Normal flora.     - Gut:         - Mechanical: Longitudinal flow of air or fluid.         - Chemical: Low pH; enzymes (pepsin); antibacterial peptides.         - Microbiological: Normal flora.     - Lungs:         - Mechanical: Movement of mucus by cilia.         - Chemical: Antibacterial peptides.     - Eyes/Nose:         - Mechanical: Tears; nasal cilia.         - Chemical: Salivary enzymes; lysozyme.

• B: Cellular Innate Defense     - Cells of Myeloid Origin:         - Monocytes/Macrophages.         - Dendritic Cells.         - Granulocytes (Neutrophils, Eosinophils, Mast cells, Basophils).     - Cells of Lymphoid Origin:         - Natural Killer (NK) cells.         - NK/T cells.

Characteristics of Innate Immune Cells

• Monocytes and Macrophages     - Action: Phagocytosis and killing of microorganisms.     - Immune Initiation: Activation of T cells and initiation of the immune response.     - Ontogeny: A monocyte is a young macrophage found in the blood; it migrates to tissues to differentiate.     - Antigen Presentation: Macrophages act as Antigen Presenting Cells (APCs).     - Cytokine Secretion: When exposed to inflammatory stimuli, they secrete cytokines including Tumor Necrosis Factor (TNF), IL-1, IL-6, IL-8, and IL-12.     - Tissue-Specific Macrophages:         - Alveolar Macrophages: Found in the lungs.         - Histiocytes: Found in connective tissue.         - Mesangial Cells: Found in the kidneys.         - Microglial Cells: Found in the central nervous system (e.g., brain).         - Kupffer Cells: Found in the liver.         - Osteoclasts: Found in the bone.

• Dendritic Cells     - Location: Found mainly in lymphoid tissue.     - Function: Function as the most potent stimulators of T-cell responses (APC function).     - Role: Activation of T cells to initiate adaptive immunity.

• Mast Cells     - Action: Expulsion of parasites through the release of granules (degranulation).     - Granule Contents: Histamine, leukotrienes, chemokines, and cytokines.     - Clinical Significance: Heavily involved in allergic responses.

• Neutrophils     - Description: Granulocytes with a multi-lobed (polymorphonuclear) nucleus.     - Granules: Contain compounds such as Defensins, lactoferrin, and MMPs (Matrix Metalloproteinases).     - Function: Professional phagocytes; critical for "clearing" bacterial infections.     - Lifespan: Very short life span, often lasting only a few hours.

• Eosinophils     - Description: Double-lobed nucleus with orange-stained granules.     - Action: Kills antibody-coated parasites through degranulation.     - Granule Contents: Toxic compounds such as Histamine and peroxide.     - Clinical Significance: Involved in allergic inflammation and fighting parasitic infections.

• Basophils     - Description: Blue granules containing toxic and inflammatory compounds.     - Granule Contents: Histamine, Heparin, and chemotactic factors.     - Action: Cell-killing cells; mild phagocytic activity.     - Clinical Significance: Important in allergic reactions; sometimes referred to as "blood Mast cells."

• Natural Killer T (NK-T) Cells     - Characteristics: Share properties of both NK cells and T cells.     - Receptors: Possess $\alpha\beta$ antigen receptors, but these lack the high diversity seen in standard T cells.     - Function: Can both suppress or activate innate and adaptive immune responses.

Adaptive Immunity

• Cells of the Adaptive Immune System     - Myeloid Origin (Supportive/APC): Monocytes, Dendritic Cells, Macrophages, Granulocytes.     - Lymphoid Origin: NK cells, NK/T cells, T Helper cells, B1 Cells.

• Lymphoid Organs     - Generative (Primary) Lymphoid Organs: Bone marrow (B cell maturation) and Thymus (T cell maturation).     - Peripheral (Secondary) Lymphoid Organs: Lymph nodes, spleen, mucosal and cutaneous lymphoid tissues.     - Recirculation: Mature lymphocytes recirculate through the blood and lymph between these organs.

• T Lymphocytes (T Cells)     - T Helper (Th) Cells:         - Markers: Express the T cell receptor (TCR) and CD4 co-receptor.         - Function: Express cytokines to coordinate the entire immune response.         - Subsets: Primarily Th1 and Th2.     - Cytotoxic T Lymphocytes (CTLs):         - Markers: Express the T cell receptor (TCR) and CD8 co-receptor.         - Function: Directly destroy infected cells.

• B Lymphocytes (B Cells)     - Programming: Each B cell is genetically programmed to produce an antibody of unique specificity.     - Diversity: The human body can theoretically produce $10^{15}$ different antibody specificities.     - Receptors: An individual B cell typically has $100,000$ identical antibody molecules on its surface acting as B cell receptors.     - Differentiation: Binding of an antigen to the B cell receptor results in differentiation into a plasma cell.

• B Cell Maturation Stages and Immunoglobulin (Ig) Production     - Stem Cell: No Ig production.     - Pre-B Cell: Produces $\mu$ heavy chain (cytoplasmic).     - Immature B Cell: Membrane IgM Expressed.     - Mature B Cell: Membrane IgM and IgD expressed.     - Activated B Cell: Low-rate Ig secretion; undergoes heavy chain isotype switching and affinity maturation.     - Plasma Cell (Antibody-secreting cell): High-rate Ig secretion; reduced levels of membrane Ig.

Core Concepts of Immune Responses

• Immune Response Defined: This is the collective and coordinated response to a foreign substance within an individual. It is mediated by the specific cells and molecules that constitute the immune system.

• The Primary Immune Response:     - This response occurs when an antigen comes into contact with the immune system for the first time ($1^{st}$ exposure).     - During this phase, the immune system must learn to recognize the specific antigen and determine how to produce antibodies against it.     - The ultimate goal of this phase is the production of memory lymphocytes.

• The Secondary Immune Response:     - This occurs during the second ($2^{nd}$), third ($3^{rd}$), fourth ($4^{th}$), or any subsequent exposure of the individual to the same antigen.     - At this stage, immunological memory has already been established.     - The immune system is capable of initiating antibody production immediately upon re-exposure.

Types of Adaptive Immune Responses

• Humoral (Antibody-Mediated) Immunity:     - This type of immunity results specifically from the presence and action of antibodies located in the blood and lymph.     - It is mediated by the $B$-cell component of the immune response.

• Cellular (Cell-Mediated) Immunity:     - This type of immunity is mediated by $T$ cells.     - Key cell types include $T$-helper ($T_H$) cells and cytotoxic $T$ lymphocytes ($CTLs$).

• Developmental Pathways of Immune Cells:     - Activated $B$ Lymphocytes: These develop into two lineages: antibody-secreting plasma cells and memory $B$ cells.     - Activated $T$ Lymphocytes: These develop into memory $T$ cells, helper $T$ cells ($T_H1$, $T_H2$, or $T_H17$), and cytotoxic $T$ cells.

Characteristics of the Primary Antibody Response

• Initial Exposure: Occurs when an individual is exposed to an antigen for the first time, whether through naturally occurring infection or through a vaccine.

• Lag Phase (Latent Period):     - There is an initial latent period of several days to weeks before a detectable antibody response is established.     - During this period, no antigen-specific antibody can be detected in the blood.     - The duration of this phase depends on the nature of the antigen, the dose administered, and the route of administration.     - Clinical Correlation: This explains why antibody-based tests, such as those for $HIV$, are not accurate until several weeks after the initial exposure.

• Antibody Sequence:     - $IgM$ is the first type of antibody secreted after $B$-cell activation during the primary exposure.

• Phase Kinetics:     - Log Phase: After the lag phase, the antibody titer rises logarithmically to a plateau.     - Affinity: The affinity of antibodies for the antigen's determinants is generally low to moderate.     - Decline Phase: The serum antibody concentration rises to a peak and then declines (decreases quickly), sometimes dropping to very low levels.     - Metabolism: During the decline phase, antibodies are naturally metabolized or cleared from circulation after binding to the antigen. $IgM$ levels tend to decline faster than $IgG$ levels.

Characteristics of the Secondary Antibody Response

• Trigger: Occurs upon secondary exposure to the same antigen (e.g., re-exposure to a pathogen or receiving a vaccine booster).

• Heightened Response: $B$-cells establish a significantly stronger and faster response.

• Antibody Class (Isotype) Switching:     - Under the influence of $T$-helper cells, the production switches from $IgM$ to other classes, most commonly $IgG$.     - Other classes produced include $IgA$ (mucosal) or $IgE$ (allergic responses).     - The new class of antibody maintains the same specificity for the original antigen as the initial $IgM$.

• Kinetics and Titer:     - Shorter Lag Phase: The latent period is much shorter ($1-4\text{ days}$) compared to the primary response.     - Higher Titer: Antibody levels rise to much higher levels ($100-1000$ times more antibodies).     - Duration: The response remains elevated for a significantly longer period.

• Affinity Maturation: The secondary response produces antibodies with a higher affinity, meaning they bind the antigen more firmly and dissociate less easily.

• Memory Cells: The secondary response is a direct result of the persistence of antigen-sensitive memory cells from the first response. These cells can persist in the body for years to provide long-term specific immunity.

Comparative Analysis: Primary vs. Secondary Immune Response

Mechanism of B-Cell Activation (Dialogue Transcription)

Below is a transcription of the interaction between immune cells during activation as depicted in the lecture materials.

• B Cell: "An invader… I must inform a $T$ cell."

• B Cell to T Cell: "Hey $T$ cell, look what I found."

• T Cell: "Hmm.. It looks non-self. Good job, $B$."

• B Cell: "YESSSS.. I knew it!"

• T Cell: "Here.. $CD40L$ \u0026 some cytokines."

• B Cell (Outcome): "I am an activated $B$ lymphocyte now."